Simultaneous determination of five active ingredients including gallic acid in different dosage forms of Sana preparation by reverse-phase high-performance liquid chromatography / 中国基层医药

Objective:To establish reverse-phase high-performance liquid chromatography and simultaneously determine gallic acid, methyl gallate, corilagin, Sennoside B, and Sennoside A levels in Sana preparations.Methods:From January to December 2021, Phenomenex Hydro-RP 80A column (4.60 mm × 250 mm, 4 μm) was used. Elution was conducted using mobile phases methanol (A)-0.2% formic acid (B). The following gradients were applied: 1%-3%A for 0-18 minutes, 3%-15%A for 18-19 minutes, 15%-17%A for 19-40 minutes, 17%-25%A for 40-45 minutes, 25%-35%A for 45-65 minutes, 35%-60%A for 65-95 minutes, 60%-90%A for 95-96 minutes, 90%-1%A for 96-97 minutes. The flow rate was 1.0 mL/minute. The column temperature was 35 ℃. The detection wavelength was 270 nm.Results:The linear ranges of gallic acid, methyl gallate, corilagin, Sennoside B and Sennoside A were 0.182-1.099 μg ( r = 0.999 9), 0.046-0.278 μg ( r = 0.999 2), 0.266-1.598 μg ( r = 0.999 4), 0.172-1.036 μg ( r = 0.999 2), and 0.176-1.056 μg ( r = 0.999 9). The average dosing recovery rates were 100.02%, 99.14%, 99.38%, 101.77%, and 100.92%, respectively. Conclusion:Reverse-phase high-performance liquid chromatography can be used for quality control of Sana preparations because of high accuracy, sensitivity, reliability, and reproduction.

Determining the Presence of 5-Fluorouracil in Hamster Saliva by HPLC / Determinação, por CLAE, da Presença do 5-Fluorouracil em Saliva de Hamsters

Abstract Introduction: Various methods of analysis for the assay of chemotherapeutic agent 5-Fluorouracil (5-FU) in human and animal biological fluids have previously been reported. However, there is no standardization for detecting 5-FU in the hamsters' saliva that received the chemotherapeutic agent. Objective: Considering that the administration of 5-FU in some way changes the morphology and function of the salivary glands, and that the presence of the chemotherapeutic agents in the oral mucosa may lead to some oral complications, the aim of this study was to determine the presence of 5-FU in the hamsters' saliva that received the chemotherapeutic agent, by means of the High Performance Liquid Chromatography technique (HPCL) since this animal model is used in studies of 5-FU induced oral mucositis and glandular hypofunction. Methods: Twelve animals were divided into 4 groups: CP and CPI, in which the animals received pilocarpine (CP) or pilocarpine + isoproterenol (CPI) and the chemotherapy vehicle intraperitoneally; and Groups QP and QPI, in which the animals received the same secretagogues listed above, and the chemotherapeutic agent 5-FU, respectively. After the secretagogue administration, saliva was collected from all the animals for a period of 60 mins. Subsequently, the saliva was frozen at -80 ˚C for later determination of the chemotherapeutic agent by HPLC. After the the chromatograms analysis, and based on the results obtained, it was possible to identify the presence of 5-FU in the saliva samples from hamsters that received the chemotherapeutic agent intraperitonally, by the HPLC technique. (AU)

Resumo Vários métodos de análise para o ensaio do quimioterápico 5-Fluorouracil (5-FU) em fluidos biológicos de humanos e animais, foram previamente relatados. No entanto, não há uma padronização para detecção de 5-FU na saliva de hamsters que receberam o quimioterápico. Considerando que a administração do 5-FU altera de alguma maneira a morfologia e função das glândulas salivares, e que a presença do quimioterápico na mucosa oral pode levar a algumas complicações orais, este trabalho teve como objetivo de determinar a presença de 5-FU na saliva de hamsters que receberam o quimioterápico pela técnica de Cromatografia Líquida de Alta Eficiência (CLAE), uma vez que este modelo animal é usado nos estudos com mucosite oral e hipofunção glandular, induzidas por 5-FU. Doze animais foram divididos em 4 grupos: CP e CPI, onde os animais receberam intraperitonealmente pilocarpina (CP) ou pilocarpina + isoproterenol (CPI) e o veículo do quimioterápico, e os grupos QP e QPI, onde os animais receberam, respectivamente, os mesmos secretagogos listados acima e o quimioterápico 5-FU. Após a administração do secretagogo, foi coletada a saliva de todos os animais, por um período de 60 min. Em seguida, a saliva foi congelada a -80 ˚C para posterior determinação do quimioterápico por CLAE. Após análise dos cromatogramas, e com base nos resultados obtidos, foi possível identificar a presença do 5-FU nas amostras de saliva de hamsters que receberam o quimioterápico via intraperitoneal pela técnica da CLAE. (AU)

Study on the relation of the radiochemical purity and in vivo imaging effect of 68Ga-DOTATATE injection / 中华核医学与分子影像杂志

Objective:To explore the relation of the radiochemical purity and in vivo imaging effect of 68Ga-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA)- D-phe1-Tyr3-Thr8-octreotide (TATE) injection. Methods:High performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) methods were established to determine 68Ga-DOTATATE, 68Ga 3+ , 68Ga in colloidal form and 68Ga-DOTA- D-Phe1-Tyr3-Thr8-dethreonine-octreotide (heptapeptide) and to study the influence of precursor purity on radiochemical purity of labelled products. The uptake of 68Ga-DOTATATE injection with different radiochemical purities was investigated in nude mice bearing AR42J cells by microPET imaging and the tumor target/non-target (T/NT) value was calculated. One-way analysis of variance and Pearson correlation analysis were used to analyze the data. Results:The contents of 68Ga 3+ and 68Ga in colloidal form were not related with precursor purity ( r values: 0.385, 0.497, P values: 0.306, 0.137), while the content of 68Ga-DOTA-heptapeptide was positively related with the purity of DOTA-heptapeptide ( r=0.957, P<0.001). The radiochemical purities of 68Ga-DOTATATE injection were (87.0±2.3)%, (86.8±0.8)% and (94.0±3.1)% when the DOTATATE purities were 90.9%, 91.6% and 99.2%, respectively. The results of microPET imaging showed that the tumor uptake was positively related with the radiochemical purity of 68Ga-DOTATATE injection ( r=0.828, P<0.001), and the T/NT values of 68Ga-DOTATATE injection with radiochemical purities of 95.7%, 85.8%, 84.5% and 79.9% were 21.25±8.84, 8.50±1.51, 11.38±1.65 and 6.01±0.99, respectively ( F=11.48, P=0.001). Conclusion:The radiochemical purity of 68Ga-DOTATATE injection is impacted by the purity of labelled precursor and manufacturing processes and is related with the imaging effect in vivo.

Effect of noise on morphological structure and functions of rat liver / 环境与职业医学

Background Noise can cause not only auditory system injury, but also liver damage. However, the biomarkers and pathological mechanism of noise-induced liver injury are not clear yet. Objective To observe the effect of noise on the morphological structure and functions of rat liver. Methods A total of 30 Wistar rats were randomly divided into a normal control group, a low noise exposure group [(95 dB sound pressure level (SPL)], and a high noise exposure group (105 dB SPL). After 30 days of noise exposure, blood was collected, and livers were harvested and fixed. The pathological changes of livers were observed. The levels of biochemical indicators of liver function, blood glucose, and blood lipid were measured. Serum metabolites were detected by ultra-high-pressure liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Differential metabolite markers and metabolic pathways were identified. Results Compared with the control group, the body weight gain decreased in the low noise group and the high noise group after noise exposure (P<0.001, P<0.05). The pathological results showed that noise caused the rat livers’ morphological and structural damage at various degrees, and damage of the high noise exposure group was more serious. Compared with the control group, the serum levels of aspartate aminotransferase, albumin, and glycosylated serum protein in the low noise exposure group were increased (P<0.05), but the total bile acid level was decreased (P<0.05). The serum levels of alanine aminotransferase, aspartate aminotransferase, albumin, triglyceride, low density lipoprotein, and glycosylated serum protein in the high noise group exposure were increased (P<0.05), but the glucose level was decreased (P<0.05). In the serum metabolomics analysis, 11 differential metabolites were screened out in the low noise exposure group, which were mainly enriched in 3 pathways (thiamine metabolism, primary bile acid biosynthesis, and bile secretion) related to liver metabolism. Four differential metabolites were screened out in the high exposure noise group, which were mainly enriched in four significantly different metabolic pathways (insulin signaling pathway, non-alcoholic fatty liver disease, bile secretion, and insulin secretion). All the metabolic pathways involved in bile acid secretion and metabolism. Conclusion Nosie exposure can not only damage the liver structure of rats, but also affects the metabolism functions of liver. The mechanism may be related to bile acid secretion metabolic pathway.

Analysis of changes in serum metabolites of uremia patients based on ultra-high performance liquid chromatography-mass spectrometry / 中华肾脏病杂志

Objective:To analyze the changes in serum metabolites of patients with uremia using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), and provide a theoretical basis for the prevention and treatment of uremia.Methods:Uremia patients from the Department of Nephrology, the Affiliated Hospital of Southwest Medical University, and the volunteers from the Health Examination Center were enrolled in this study. According to the inclusion and exclusion criteria, 20 uremia patients (experimental group) and 20 volunteers (control group) were screened out. UHPLC-MS was used to detect the metabolites in the serum of subjects from the two groups, and difference analysis was made to screen the different metabolites, followed by correlation analysis and pathway enrichment study.Results:A total of 412 metabolites were identified by UHPLC-MS. Principal components analysis (PCA) proved that these metabolites could distinguish the control group and the experimental group well. The criteria [variable importance for the projection (VIP)>1, fold changes (FC)>1.25 or FC<0.8 and P value<0.05] was set to screen those significantly different metabolites. Finally, 28 significantly different metabolites were screened out, of which 18 metabolites increased significantly, the other 10 different metabolites decreased significantly. Correlation analysis results proved a certain correlation among 28 different metabolites and the experimental group and control group samples, and between the 28 differential metabolites themselves. Enrichment analysis found that 28 different metabolites might enrich the catecholamine biosynthetic pathway, and pathway analysis suggested that 28 different metabolites might affect glutamate, aspartame acid and glutamate metabolic pathways. Conclusion:Based on metabonomic analysis, some metabolites in the serum of patients with uremia have changed, which can affect some metabolic pathways, thus affecting the pathophysiological process of patients with uremia.

Phytochemical characterization, and antioxidant and antibacterial activities of the hydroethanolic extract of Anadenanthera peregrina stem bark / Caracterização fitoquímica, atividade antioxidante e antibacteriana do extrato hidroetanólico de cascas do caule de Anadenanthera peregrina

The Brazilian Cerrado biome consists of a great variety of endemic species with several bioactive compounds, and Anadenanthera peregrina (L.) Speg is a promising species. In this study, we aimed to perform phytochemical characterization and evaluate the antioxidant and antibacterial activities against Staphylococcus aureus and Escherichia coli of the hydroethanolic extract of A. peregrina stem bark. The barks were collected in the Botanical Garden of Goiânia, Brazil. The hydroethanolic extract was obtained by percolation and subjected to physicochemical screening, total phenolic content estimation, high-performance liquid chromatography (HPLC) fingerprinting, and antioxidant (IC50 values were calculated for the 2,2-diphenyl-1-picrylhydrazyl assay - DPPH) and antibacterial activity determination. The pH of the extract was 5.21 and density was 0.956 g/cm3. The phytochemical screening indicated the presence of cardiac glycosides, organic acids, reducing sugars, hemolytic saponins, phenols, coumarins, condensed tannins, flavonoids, catechins, depsides, and depsidones derived from benzoquinones. The extract showed intense hemolytic activity. The total phenolic content was 6.40 g GAE 100 g-1. The HPLC fingerprinting analysis revealed the presence of gallic acid, catechin, and epicatechin. We confirmed the antioxidant activity of the extract. Furthermore, the extract did not inhibit the growth of E. coli colonies at any volume tested, but there were halos around S. aureus colonies at all three volumes tested. These results contribute to a better understanding of the chemical composition of A. peregrina stem bark and further support the medicinal applications of this species.

O bioma Cerrado brasileiro apresenta em uma grande variedade de espécies endêmicas com diversos compostos bioativos, e Anadenanthera peregrina (L.) Speg é uma espécie promissora. Neste estudo, objetivamos realizar a caracterização fitoquímica e avaliar as atividades antioxidantes e antibacterianas contra Staphylococcus aureus e Escherichia coli do extrato hidroetanólico de cascas do caule de A. peregrina. As cascas foram coletadas no Jardim Botânico de Goiânia, Brasil. O extrato hidroetanólico foi obtido por percolação e submetido a triagem físicoquímica, estimativa de conteúdo fenólico total, impressão digital por cromatografia líquida de alta eficiência (HPLC) e determinação da atividade antioxidante (valores de IC50 foram calculados para o ensaio 2,2-difenil-1-picril-hidrazil) e antibacteriana. O pH do extrato foi de 5,21 e a densidade foi de 0,956 g/cm3. A triagem fitoquímica indicou a presença de glicosídeos cardíacos, ácidos orgânicos, açúcares redutores, saponinas hemolíticas, fenóis, cumarinas, taninos condensados, flavonóides, catequinas, depsídios e depsidonas derivados de benzoquinonas. O extrato mostrou intensa atividade hemolítica. O conteúdo fenólico total foi de 6,40 g de GAE 100 g-1. A análise por impressão digital por HPLC revelou a presença de ácido gálico, catequina e epicatequina. Confirmamos a atividade antioxidante do extrato. Além disso, o extrato não inibiu o crescimento de colônias de E. coli em nenhum volume testado, mas houve halos em torno das colônias de S. aureus nos três volumes testados. Estes resultados contribuem para uma melhor compreensão da composição química da casca de A. peregrina e apoia ainda mais as aplicações medicinais desta espécie.

Phytochemical characterization, and antioxidant and antibacterial activities of the hydroethanolic extract of Anadenanthera peregrina stem bark / Caracterização fitoquímica, atividade antioxidante e antibacteriana do extrato hidroetanólico de cascas do caule de Anadenanthera peregrina

The Brazilian Cerrado biome consists of a great variety of endemic species with several bioactive compounds, and Anadenanthera peregrina (L.) Speg is a promising species. In this study, we aimed to perform phytochemical characterization and evaluate the antioxidant and antibacterial activities against Staphylococcus aureus and Escherichia coli of the hydroethanolic extract of A. peregrina stem bark. The barks were collected in the Botanical Garden of Goiânia, Brazil. The hydroethanolic extract was obtained by percolation and subjected to physicochemical screening, total phenolic content estimation, high-performance liquid chromatography (HPLC) fingerprinting, and antioxidant (IC50 values were calculated for the 2,2-diphenyl-1-picrylhydrazyl assay - DPPH) and antibacterial activity determination. The pH of the extract was 5.21 and density was 0.956 g/cm3. The phytochemical screening indicated the presence of cardiac glycosides, organic acids, reducing sugars, hemolytic saponins, phenols, coumarins, condensed tannins, flavonoids, catechins, depsides, and depsidones derived from benzoquinones. The extract showed intense hemolytic activity. The total phenolic content was 6.40 g GAE 100 g-¹. The HPLC fingerprinting analysis revealed the presence of gallic acid, catechin, and epicatechin. We confirmed the antioxidant activity of the extract. Furthermore, the extract did not inhibit the growth of E. coli colonies at any volume tested, but there were halos around S. aureus colonies at all three volumes tested. These results contribute to a better understanding of the chemical composition of A. peregrina stem bark and further support the medicinal applications of this species.

O bioma Cerrado brasileiro apresenta em uma grande variedade de espécies endêmicas com diversos compostos bioativos, e Anadenanthera peregrina (L.) Speg é uma espécie promissora. Neste estudo, objetivamos realizar a caracterização fitoquímica e avaliar as atividades antioxidantes e antibacterianas contra Staphylococcus aureus e Escherichia coli do extrato hidroetanólico de cascas do caule de A. peregrina. As cascas foram coletadas no Jardim Botânico de Goiânia, Brasil. O extrato hidroetanólico foi obtido por percolação e submetido a triagem físico química, estimativa de conteúdo fenólico total, impressão digital por cromatografia líquida de alta eficiência(HPLC) e determinação da atividade antioxidante (valores de IC50 foram calculados para o ensaio 2,2-difenil-1-picril-hidrazil) e antibacteriana. O pH do extrato foi de 5,21 e a densidade foi de 0,956 g/cm3. A triagem fitoquímica indicou a presença de glicosídeos cardíacos, ácidos orgânicos, açúcares redutores, saponinas hemolíticas, fenóis, cumarinas, taninos condensados, flavonóides, catequinas, depsídios e depsidonas derivados de benzoquinonas. O extrato mostrou intensa atividade hemolítica. O conteúdo fenólico total foi de 6,40 g de GAE 100 g-1. A análise por impressão digital por HPLC revelou a presença de ácido gálico, catequina e epicatequina. Confirmamos a atividade antioxidante do extrato. Além disso, o extrato não inibiu o crescimento de colônias de E. coli em nenhum volume testado, mas houve halos em torno das colônias de S. aureus nos três volumes testados. Estes resultados contribuem para uma melhor compreensão da composição química da casca de A. peregrina e apoia ainda mais as aplicações medicinais desta espécie.

Phytochemical characterization, and antioxidant and antibacterial activities of the hydroethanolic extract of Anadenanthera peregrina stem bark

Abstract The Brazilian Cerrado biome consists of a great variety of endemic species with several bioactive compounds, and Anadenanthera peregrina (L.) Speg is a promising species. In this study, we aimed to perform phytochemical characterization and evaluate the antioxidant and antibacterial activities against Staphylococcus aureus and Escherichia coli of the hydroethanolic extract of A. peregrina stem bark. The barks were collected in the Botanical Garden of Goiânia, Brazil. The hydroethanolic extract was obtained by percolation and subjected to physicochemical screening, total phenolic content estimation, high-performance liquid chromatography (HPLC) fingerprinting, and antioxidant (IC50 values were calculated for the 2,2-diphenyl-1-picrylhydrazyl assay - DPPH) and antibacterial activity determination. The pH of the extract was 5.21 and density was 0.956 g/cm3. The phytochemical screening indicated the presence of cardiac glycosides, organic acids, reducing sugars, hemolytic saponins, phenols, coumarins, condensed tannins, flavonoids, catechins, depsides, and depsidones derived from benzoquinones. The extract showed intense hemolytic activity. The total phenolic content was 6.40 g GAE 100 g-1. The HPLC fingerprinting analysis revealed the presence of gallic acid, catechin, and epicatechin. We confirmed the antioxidant activity of the extract. Furthermore, the extract did not inhibit the growth of E. coli colonies at any volume tested, but there were halos around S. aureus colonies at all three volumes tested. These results contribute to a better understanding of the chemical composition of A. peregrina stem bark and further support the medicinal applications of this species.

Resumo O bioma Cerrado brasileiro apresenta em uma grande variedade de espécies endêmicas com diversos compostos bioativos, e Anadenanthera peregrina (L.) Speg é uma espécie promissora. Neste estudo, objetivamos realizar a caracterização fitoquímica e avaliar as atividades antioxidantes e antibacterianas contra Staphylococcus aureus e Escherichia coli do extrato hidroetanólico de cascas do caule de A. peregrina. As cascas foram coletadas no Jardim Botânico de Goiânia, Brasil. O extrato hidroetanólico foi obtido por percolação e submetido a triagem físico-química, estimativa de conteúdo fenólico total, impressão digital por cromatografia líquida de alta eficiência (HPLC) e determinação da atividade antioxidante (valores de IC50 foram calculados para o ensaio 2,2-difenil-1-picril-hidrazil) e antibacteriana. O pH do extrato foi de 5,21 e a densidade foi de 0,956 g/cm3. A triagem fitoquímica indicou a presença de glicosídeos cardíacos, ácidos orgânicos, açúcares redutores, saponinas hemolíticas, fenóis, cumarinas, taninos condensados, flavonóides, catequinas, depsídios e depsidonas derivados de benzoquinonas. O extrato mostrou intensa atividade hemolítica. O conteúdo fenólico total foi de 6,40 g de GAE 100 g-1. A análise por impressão digital por HPLC revelou a presença de ácido gálico, catequina e epicatequina. Confirmamos a atividade antioxidante do extrato. Além disso, o extrato não inibiu o crescimento de colônias de E. coli em nenhum volume testado, mas houve halos em torno das colônias de S. aureus nos três volumes testados. Estes resultados contribuem para uma melhor compreensão da composição química da casca de A. peregrina e apoia ainda mais as aplicações medicinais desta espécie.

Quantitative analysis of active ingredient content of Rhodiola rosea at different harvest months / 国际中医中药杂志

Objective:To establish a method for quantitative analysis of the active ingredients including salidroside, rosarin and rosavin and content determination in Rhodiola rosea at different harvest months. Methods:HPLC was used on an X selectHSS T3 (250 mm×4.6 mm, 5 μm) column with mobile phase consisting of methol-acetonitrile-phosphoric acid (0.05%) aqueous solution for gradient elution at a flow rate of 1 ml/min. The wavelength was detected at 275 nm (salidroside) and 254 nm (rosarin, rosavin). The column temperature was set at 30 ℃ and the injection volume was 5 μl.Results:The peak areas of Salidroside, rosarin and rosavin showed good linear relationships ( r > 0.999) with the content in the ranges of 44-1 420, 10-307 and 18-573 μg, respectively. The method was precise, stable, repeatable and the sample recovery test all well satisfied the requirements of quantitative analysis. The highest accumulation of the active ingredients was observed in Rhodiola rosea in September and the content of salidroside, rosarin and rosavin were 0.66, 0.07 and 0.53 mg/g, respectively. Conclusion:This method is simple and rapid to evaluate the content of active ingredients in Rhodiola rosea.

Research of Shangke Huoxue Decoction based on multi-component TLC identification and content determination / 国际中医中药杂志

Objective:To establish the TLC identification method and content determination method of ferulic acid, ligustilide, hydroxysafflor yellow A and paeoniflorin in Shangke Huoxue Decoction for quality evaluation.Methods:Ferulic acid, ligustilide, hydroxysafflor yellow A and paeoniflorin were qualitatively identified by TLC method, and the content was determined by HPLC method. Waters Symmetry ShieldTM RP18 column (4.6 mm×250 mm, 5 μm) was set, the mobile phase consisted of acetonitrile-0.15% phosphoric acid water with gradient elution at a flow of 1.0 ml/min, and the column temperature was 30 ℃.The detection wavelength was 320 nm (33-50 min for ferulic acid, 55-70 min for ligustilide), 403 nm (7-31 min for hydroxysafflor yellow A) and 230 nm (7-31 min for paeoniflorin).Results:The TLC spots were clear. The linear relationships of ferulic acid, ligustilide, and hydroxysafflor yellow A were good in the range of 3.05-48.74 μg, 3.50-26.24 μg, 21.34-213.44 μg. The method was stable, repeatable with good recovery rate.Conclusion:The TLC and HPLC method for the simutanous determination of the four effective components in Shangke Huoxue Decoction were established, and the methods are suitable for the quality evaluation of Shangke Huoxue Decoction.

Validation of a method employing liquid chromatography to identify and quantify glycomacropeptide of casein in raw milk / Validación de un método mediante cromatografía líquida para identificar y cuantificar glicomacropéptido de caseína en la leche cruda

Background: The information on official testing methods, or regulatory methods in Colombia to test whey in milk is limited; this restriction of information goes against the possibility of mitigating the risk of food fraud. Objectives: The validation of an HPLC method to determine casein glycomacropeptide (c-GMP), a protein that countries such as Brazil, Spain, and Ecuador have used as an indicator of raw milk adulteration with whey, was carried out. Methods: A 10mL sample of raw milk is precipitated with 24% TCA using ultrasound, a process followed by filtration. The collected fraction ensured the separation of c-GMP and then injected into the liquid chromatography. Results: A 30 minutes analysis allowed the determination of c-GMP with a retention time of 12.9 ± 0.5 minutes. The performance characteristics method in the validation exercise were: recovery percentage 99.97%, linearity R2> 0.95; % RSD accuracy <5.3%. Conclusion, the method exhibits desirable attributes for the intended purpose

Antecedentes: En Colombia la información de dominio público en metodologías de análisis de lactosuero en leche es limitada, restringiendo la posibilidad de acceder a ellas para mitigar el riesgo de fraude alimentario. Objetivos: Se realizó validación de un método por HPLC para determinar en leche cruda c-GMP, proteína usada como indicador de adulteración en países como Brasil y Ecuador. Metodos: Una muestra de 10mL de leche cruda es precipitada con TCA al 24% empleando ultrasonido, proceso seguido por filtración. La fracción recolectada aseguró la separación del c-GMP para luego inyectar al cromatógrafo líquido. Resultados: La determinación de c-GMP permitió el análisis en 30 minutos con tiempo de retención de 12,9 ± 0,5 minutos. Las características de desempeño del método en el ejercicio de validación fueron: porcentaje de recuperación 99,97%, linealidad R2>0,95; precisión %RSD< 5,3%. Conclusión: el método al final del ejercicio exhibe atributos para el fin previsto

High peak of carbamylated hemoglobina discordant with urea level: a case report / Pico alto da hemoglobina carbamilada discordante com o nível de ureia: relato de caso

ABSTRACT Hemoglobin A1c (HbA1c) measurement is commonly performed in diabetes mellitus patients to monitor glycemic control over the last three to four months. Carbamylated hemoglobin, which is the hemoglobin that binds to isocyanic acid derived from urea, is one of the possible analytical interference in the uremic patient. When measured by ion-exchange high-performance liquid chromatography (HPLC), carbamylated hemoglobin forms a peak that overlaps the peak of HbA1c, causing a falsely elevated HbA1c result. We report a case of a 60-years-old man who had a spurious increase in HbA1c, with a high carbamylated hemoglobin peak disproportionate to the urea value. Subsequent hemoglobin analysis using hemoglobin electrophoresis and HPLC hemoglobin testing system suggested hemoglobin J (Hb J) variant. Our case highlighted the possibility of misleading HbA1c interpretation in the presence of a high carbamylated hemoglobin peak, but not proportionate to urea value. In this study, Hb J was detected. A method free from hemoglobin variant interference should be used ideally, and monitoring glycemic control should be performed using alternative methods, such as serum fructosamine or continuous glucose monitoring.

RESUMEN La prueba de la hemoglobina glicosilada A1c (HbA1c) se realiza comúnmente en pacientes con diabetes mellitus para monitorear el control glucémico durante los últimos tres o cuatro meses. La hemoglobina carbamilada, que es la hemoglobina que se une al ácido isociánico derivado de la urea, es una de las posibles interferencias analíticas en el paciente urémico. Cuando se mide mediante cromatografía líquida de alta eficacia (HPLC) de intercambio iónico, la hemoglobina carbamilada forma un pico que se superpone al pico de HbA1c, lo que provoca un resultado de HbA1c falsamente elevado. Presentamos el caso de un hombre de 60 años que tuvo un aumento espurio de HbA1c, con un pico de hemoglobina carbamilada alto desproporcionado al valor de urea. El análisis de hemoglobina posterior mediante electroforesis de hemoglobina y el sistema de prueba de hemoglobina HPLC sugirió una variante de hemoglobina J (Hb J). Nuestro caso destacó la posibilidad de una interpretación engañosa de la HbA1c en presencia de un pico de hemoglobina carbamilada alto, pero no proporcional al valor de urea. En este estudio, se detectó Hb J. Lo ideal sería utilizar un método libre de interferencia de variantes de hemoglobina, y la monitorización del control glucémico debería realizarse mediante métodos alternativos, como la fructosamina sérica o la monitorización continua de la glucosa.

RESUMO A dosagem de hemoglobina A1c (HbA1c) é comumente realizada em pacientes com diabetes mellitus para monitorar o controle glicêmico nos últimos três a quatro meses. A hemoglobina carbamilada - hemoglobina que se liga ao ácido isociânico derivado da ureia - é uma das possíveis interferências analíticas no paciente urêmico. Quando medida por cromatografia líquida de alta eficiência (HPLC) por troca iônica, a hemoglobina carbamilada forma um pico que se sobrepõe ao pico da HbA1c, causando um resultado falsamente elevado da HbA1c. Relatamos o caso de um homem de 60 anos de idade que apresentava um aumento espúrio de HbA1c, com alto pico de hemoglobina carbamilada desproporcional ao valor de ureia. A análise subsequente da hemoglobina usando eletroforese de hemoglobina e sistema de teste de hemoglobina por HPLC sugeriu a variante de hemoglobina J (Hb J). Nosso caso destacou a possibilidade de interpretação enganosa da HbA1c na presença de um pico alto de hemoglobina carbamilada, mas não proporcional ao valor da ureia. Neste estudo, foi detectada a Hb J. Um método isento de interferência de variantes da hemoglobina deve ser idealmente usado, e o monitoramento do controle glicêmico deve ser feito usando métodos alternativos, como frutosamina sérica ou monitoramento contínuo da glicose.

Detección de variantes de hemoglobina en pacientes examinados por hemoglobina A1c / Detection of hemoglobin variants in patients tested for Hemoglobin A1c

Resumen Justificación: La genética en las variantes de hemoglobina en Costa Rica es resultado del cruce de caracteres autóctonos indígenas con poblaciones inmigrantes de europeos, africanos y otros, desde el periodo de la Conquista, que contribuyeron a la mezcla genética presente en la población de Costa Rica. Las hemoglobinopatías mayormente distribuidas en la población humana son: hemoglobina S, C, D y E, siendo la hemoglobina S la más frecuente y la que presenta consecuencias más graves. Objetivo: Detectar variantes de hemoglobina en pacientes examinados por hemoglobina A1c, en la sección de Química Clínica del laboratorio de la Clínica de Filadelfia de la Caja Costarricense de Seguro Social, en el Cantón de Carrillo, Guanacaste, Costa Rica, durante el período de enero a octubre de 2018. Métodos: Se analizaron 2775 muestras sanguíneas de pacientes de los nueve equipos básicos de salud que conforman el Área de Salud de Carrillo, y que además requieren estudio por hemoglobina glicosilada en el período de enero a octubre de 2018. El análisis se realizó en el Laboratorio del Área de Salud de Carrillo. Las muestras fueron recolectadas en tubos vacutainer con EDTA y analizadas en el equipo automatizado TOSOH HLC-723GX, utilizando la metodología HPLC cromatografía de intercambio catiónico con la separación y cuantificación de las diferentes fracciones de hemoglobina. Los datos se analizaron en plantilla de Microsoft Excel. Resultados: En 2775 pacientes examinados por hemoglobina A1c, 167 (6,0 %) fueron portadores de variantes de hemoglobina, con una frecuencia de 1/17, en donde el 97 % correspondió a heterocigotos para hemoglobina un 3% a heterocigotos para hemoglobina C y ninguno para la variante D. La presencia de variantes se observó en los 9 equipos básicos de atención integral en salud del área. La distribución de portadores por equipo básico de atención en el Área de Salud de Carrillo varió de un 4,0 % a un 9,3 %. Conclusiones: Un 6 % de las muestras analizadas presentó variantes de hemoglobina, siendo la hemoglobina S la predominante. Esta característica presente en la población del cantón de Carrillo merece atención a nivel de salud pública; la metodología existente a nivel de área permite estudiar a un grupo de población (costo efectivo) en riesgo que precisa vigilancia y asesoramiento genético, con el fin de concienciar a la población respecto al problema, reducir la incidencia de la enfermedad y prolongar la supervivencia de los afectados.

Abstract Background: The genetics in hemoglobin variants in Costa Rica are a result of the crossing of autochthonous indigenous characters with European, African and other immigrant populations. All of these contributed to the genetic mixture that is currently present in Costa Rica's population. The most distributed hemoglobinopathies in the human population are: hemoglobin S, C, D, and E, with hemoglobin S being the most frequent and having the most serious consequences. Objective: Detection of hemoglobin variants in patients who were examined for hemoglobin A1c in the Clinical Chemistry section of the Filadelfia Clinic Laboratory, from January to October 2018. The clinic is in Canton of Carrillo, Guanacaste (Costa Rica), and it is part of the social security system. Methods: 2775 blood samples and their respective data were analyzed from patients of the nine basic health teams that make up the Carrillo Health Area and required a study for glycosylated hemoglobin from January to October 2018. The analysis was performed in the Carrillo Health Area Laboratory. The samples were collected in vacutainer tubes containing EDTA and analyzed in the TOSOH HLC-723GX automated equipment, using the HPLC cation exchange chromatography methodology with the separation and quantification of the different hemoglobin fractions. The data was then analyzed in a Microsoft Excel template. Results: In the 2775 patients examined for hemoglobin A1c, 167 (6.0%) were found to be carriers of hemoglobin variants, with a frequency of 1/17, where 97% corresponded to heterozygotes for hemoglobin S, 3% heterozygous for hemoglobin C, and none for variant D. The presence of variants was observed in the 9 basic teams of integral health care of the area, and the distribution varied from 4% to 9,3% between them. Conclusions: A total of 6% of the samples analyzed showed a hemoglobin variant, being hemoglobin S the most predominant. This characteristic present in the population of Canton of Carrillo deserves attention at the public health level. The existing methodology at the level area allows professionals to study a population group at risk that deserves surveillance and genetic counseling, in order to raise awareness about the problem, reduce the incidence of the disease, and prolong the survival of those affected by it.

Ultra-high performance liquid chromatography tandem mass spectrometry method for detection of α-hydroxybutyrate / 中华检验医学杂志

Objective To establish an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for detecting α-hydroxybutyrate (α-HB) in serum.Methods Electrospray ionization negative ion and multiple reaction monitoring mode were used to detect serum α-HB.The linearity,low limits of quantification,precision,recovery and interference of UHPLC-MS/MS were evaluated.The reference interval of this method was established in 130 serum samples (62 males and 68 females) from Shanghai East Hospital.Dixon method was used to judge the outliers and K-S test was used to analyze the data normality.The standard curve was scored by linear regression analysis.Results The total run time was 4 min of UHPLC-MS/MS method for the determination of α-HB.It has a good linear relationship in the range of 0.5-40.0 mg/L(r=0.999 4);the low limit of quantification was 0.5 mg/L;the in-batch and inter-batch coefficient of variation precision were less than 4.1％ and 6.3％,respectively;the recovery ranged between 95.8％ and 103.8％.Hemolytic samples (about 5 g/L hemoglobin),lipemic samples (about 12 mmol/L triglyceride),icteric samples (about 150 μmol/L total bilirubin) had no significant interference to the detection.The reference range of the apparent healthy population was 1.46-6.48 mg/L.Conclusions A method for the determination of serum α-HB by UHPLC-MS/MS was established.The method was simple,rapid,and could be used for the detection of clinical samples.

Comparison between the triglycerides determination by HPLC in China and reference procedure used in the isotope dilution mass spectrometry CDC CRMLN / 中华检验医学杂志

Objective To evaluate the comparability and consistency of two kinds of triglycerides reference methods, one of which is the isotope dilution liquid chromatography-mass spectrometry (LC/MS) in the Cholesterol Reference Method Laboratory Network (CRMLN), the other isthehigh-performance liquid chromatography (HPLC) method for triglyceride detection in China. Methods 52 fresh frozen sera with triglycerides levels among 0.45-4.52 mmol/L were determined by LC/MS and HPLC. After evaluation the precision and accuracy of the two methods,a series of analyses were conducted including plotting to scatter plots and deviation graphs, testing outliers, selecting the best fitting regression models and calculating the regression equations and parameters, and so on. The expected deviation at the level of medical decision of triglycerides and the 95％confidence range were statistically analyzed.Results For HPLC method, the CV of instrument measurement was 0.29％(0％-1.16％), the total CV of samples measurement was 0.54％(0.04％-1.28％), and the average bias of the reference materials was 0.22％(-0.43％-0.68％). ForLC/MSmethod,the CV of instrument measurement was 0.55％(0％-1.68％),the total CV of samples measurement was 0.79％(0％-1.93％), and the average bias of the NIST reference materials was 0.09％(-0.73％-1.29％). No outlier was found from the scatter plots and the statistical analysis and the linear regression was fitted to analyze the results of the two methods. The linear regression parameters of two methods for 52 fresh frozen human sera were as follows:the slope was 0.9988,the standard error of slope was 0.0035, the intercept was 0.0037mmol/L, the standard error of intercept was 0.0030 mmol/L, the standard error of Y-estimate was 0.0236 mmol/L,and the correlation coefficient was 0.9997. Compared with the LC/MS method,the absolute deviation of fresh sera by HPLC method was-0.001 mmol/L (-0.070-0.056 mmol/L), with a relative deviation of 0.13％ (-2.01-2.83％). T-test showed no statistically significant difference between the two methods. The expected deviations at the triglycerides medicine decision level were within the 95％confidence range,and the expected deviations were far less than the allowable error. Conclusions The HPLC method of triglyceridesdetetion has good consistency and comparability with LC/MS method as one of the reference methods of CRMLN. Because of the advantages of HPLC method such as low cost, simplicity,less technical need,and better precision,HPLC method is expected to play an important role in the process of standardization and traceability of serum triglycerides.

Simultaneous determination of anti-diabetic drugs

A novel reverse phase, isocratic HPLC method is described to separate five anti-diabetic drugs i.e., glimepiride, metformin, sitagliptin, rosiglitazone and pioglitazone. Nucleosil C18 analytical column was used as stationary phase, while mobile phase consisted of acetonitrile:phosphate buffer: methanol (40/40/20, v/v) pH 2.0. Effluent was monitored at a flow rate 1 mL/min and detected at wavelength of 240 nm. This research produced excellent chromatography over a wide concentration range of 25-10000 ng/mL. Sepprated and well resolved quantifiable peaks were obtained and test results were linear in this range. Correlation coefficient of more than 0.9990 was witnessed as well as Low %RSD values i.e., maximum 2.0% documented excellent precision of the method. Good recoveries from pharmaecutical (99-101%), urine and plasma samples (>96%) in a range of concentrtion granted very good linearity, accuracy and precision. The projected method has satisfactory applications in quality control of these molecules as well as quantification of these molecules in urine and plasma samples.

Application of quality by design approach in RP-HPLC method development for simultaneous estimation of saxagliptin and dapagliflozin in tablet dosage form

A simple, sensitive, precise, accurate and robust high performance liquid chromatographic method has been developed for simultaneous estimation of saxagliptin (SAXA) and dapagliflozin (DAPA) in pharmaceutical formulation. Design of experiments (DoE) was applied for multivariate optimization of the experimental conditions of RP-HPLC method. Risk assessment was performed to identify the critical method parameters. Three independent factors; mobile phase composition, flow rate and column temperature were used to design mathematical models. Central composite design (CCD) was used to study the response surface methodology and to study in depth the effects of these independent factors. Desirability function was used to simultaneously optimize the retention time and resolution of SAXA and DAPA. The optimized and predicted data from contour diagram consisted of acetonitrile and ortho phosphoric acid (0.1%) in the ratio of 50:50 respectively, at a flow rate of 0.98 ml/min and column temperature 31.4 °C. Using these optimum conditions baseline separation of both drugs with good resolution and run time of less than 6 min were achieved. The optimized assay conditions were validated according to ICH guidelines. Hence, the results clearly showed that Quality by design approach could be successfully applied to optimize RP-HPLC method for simultaneous estimation of SAXA and DAPA.

Validated analytical study of the effect of Lycopene on the pharmacokinetics of Paracetamol and Chlorzoxazone in rats

Lycopene was reported to influence some cytochrome P450 enzymes activity. The present study investigates the effect of lycopene on the pharmacokinetics of paracetamol and chlorzoxazone. Lycopene (20 mg/kg) was intra-peritoneally administered to two groups of rats for eight consecutive days and two other groups were given vehicle. On the eighth day, chlorzoxazone and paracetamol were separately intravenously administered to a lycopene group and a control group. Blood samples were collected at different time intervals, treated and analyzed using HPLC. The HPLC method used for paracetamol analysis was based on isocratic elution using a mobile phase consisting of water: methanol, (77:23 v/v) at a flow rate 1 mL min−1, Kromasil C18 column, and UV detection at 254 nm using caffeine as internal standard. About chlorzoxazone, separation was carried out using water: acetonitrile (60: 40, v/v) as the mobile phase at a flow rate 1 mL min−1, Inertsil ODS-3 C18 column, UV detection at 283 nm and esomeprazole as internal standard. Statistical analysis of the pharmacokinetic data using student t test showed a significant increase in AUC 0-t , AUC 0-Inf and t1/2 of paracetamol (P<0.05) and of chlorzoxazone (P<0.05) in the groups pretreated with lycopene (20 mg/kg), significant increase in the volume of distribution of paracetamol (P < 0.05), but no significant difference in that of chlorzoxazone. In other words, paracetamol and chlorzoxazone showed significant decrease (P < 0.05), respectively. These results demonstrate that treatment of rats with Lycopene (20mg/kg, ip) has a significant effect on the metabolic clearance and the pharmacokinetics of both drugs

Degradação do repelente DEET pelas lacases do Pleurotus ostreatus / Degradation of repellent DEET by Pleurotus ostreatus laccases

Objetivo: Avaliar a capacidade de remediação do DEET em meio líquido, pelo fungo de decomposição branca Pleurotus ostreatus usando como indutor enzimático os resíduos sólidos do cacau e realizar bioensaios de toxicidade com as amostras pós-tratamento, para aplicações em tratamentos de águas. Método: Foi realizada a produção enzimática com resíduos do Cacau. A biorremediação com o caldo enzimático foi realizada em erlenmeyers de 250mL, contendo a solução do composto, tampão acetato de sódio pH 5 e o caldo batata, incubados à 28°C, com rotação de 120 rpm, por 48 horas. Já com o fungo ativo, o mesmo foi incubado a 28 ºC e teve em seu meio a adição do composto. As amostras foram quantificados em Cromatografia líquida de alta performance (CLAE). O teste de adsorção foi feito com o fungo autoclavado e analisado após 14 dias. Resultado: O composto se apresentou possivelmente tóxico e a remediação mostrou uma tendência linear de degradação com o fungo de 39%. Conclusão: Pleurotus ostreatus é um candidato promissor para o tratamento de contimanações geradas por DEET.

Objective: We evaluated the remediation capacity of DEET in liquid medium by the white decomposition fungus Pleurotus ostreatus using the solid residues of cocoa as an enzymatic inducer and performed toxicity bioassays with the post-treatment samples, for water treatment applications. Method: Enzymatic production with cocoa residues was performed. Bioremediation with the enzyme broth was performed in a 250mL erlenmeyer flasks, containing the solution of the compound, sodium acetate buffer pH 5 and the potato broth, incubated at 28 °C, with rotation of 120 rpm, for 48 hours. With the active fungus, the same was incubated at 28 ºC and had in its culture medium the addition of the compound. The samples were quantified in high performance liquid chromatography (HPLC). The adsorption test was performed with the autoclaved fungus and analyzed after 14 days. Results: The compound was possibly toxic and the remediation showed a linear tendency of degradation of 39% with the fungus. Conclusion: Pleurotus ostreatus is a promising candidate for the treatment of contaminants generated by DEET.

Determination of genistin and genistein contents in flemingia philippinensis by HPLC / 国际中医中药杂志

Objective To determine the contents of genistin and genistein in flemingia philippinensis by HPLC. Methods The contents of genistin and genistein in flemingia philippinensis were quantitatively determined by HPLC method. Results The linear relationship between genistin and genistein was good (r=0.9999, RSD=1.77％, 1.16％). There were differences in the contents of genistin and genistein in flemingia philippinensis of different regions, and the genistin content in Yangshuo city of Guangxi was the highest (0.575 mg/g), while the genistein content in Fuzhou city of Guangxi was the highest (0.264 mg/g). Conclusions The HPLC method has good reproducibility and stability in measuring the contents of genistin and genistein in flemingia philippinensis. The contents of genistin and genistein are different in flemingia philippinensis from different areas in Guangxi.